Platelet α-granule cargo packaging and release are affected by the luminal proteoglycan, serglycin

Background Serglycin (SRGN) is an intragranular, sulfated proteoglycan in hematopoietic cells that affects granule composition and function. Objective To understand how SRGN affects platelet granule packaging, cargo release, and extra-platelet microenvironments. Methods Platelets and megakaryocytes from SRGN(-/-) mice were assayed for secretion kinetics, cargo levels, granule morphology upon activation, and receptor shedding. Results Metabolic, (SO4)-S-35 labeling identified SRGN as a major sulfated macromolecule in megakaryocytes. SRGN colocalized with alpha-granule markers (platelet factor 4 [PF4], von Willebrand factor [VWF], and P-selectin), but its deletion did not affect alpha-granule morphology or number. Platelet alpha-granule composition was altered, with a reduction in basic proteins (pI >= 8; e.g., PF4, SDF-1, angiogenin) and constitutive release of PF4 from SRGN(-/-) megakaryocytes. P-Selectin, VWF, and fibrinogen were unaffected. Serotonin (5-HT) uptake and beta-hexosaminidase (HEXB) were slightly elevated. Thrombin-induced exocytosis of PF4 from platelets was defective; however, release of RANTES/CCL5 was normal and osteopontin secretion was more rapid. Release of 5-HT and HEXB (from dense granules and lysosomes, respectively) were unaffected. Ultrastructural studies showed distinct morphologies in activated platelets. The alpha-granule lumen of SRGN(-/-) platelet had a grainy staining pattern, whereas that of wild-type granules had only fibrous material remaining. alpha-Granule swelling and decondensation were reduced in SRGN(-/-) platelets. Upon stimulation of platelets, a SRGN/PF4 complex was released in a time- and agonist-dependent manner. Shedding of GPVI from SRGN(-/-) platelets was modestly enhanced. Shedding of GP1b was unaffected. Conclusion The polyanionic proteoglycan SRGN influences alpha-granule packaging, cargo release, and shedding of platelet membrane proteins.